Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3311. Adviser: Marc I. Diamond.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3337. Advisers: Graeme W. Davis; Robert H. Edwards.

Thesis (Ph.D.)--Brown University, 2008. / Advisor : Mark F. Bear. Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2108. Adviser: Roger Nicoll.

<p> Life exists in complex environments, requiring organisms to have adaptive developmental strategies in order to survive. The nematode <i>Caenorhabditis elegans</i> has evolved a developmental polyphenism whereby it may arrest development in the dauer diapause state when environmental conditions are not suitable for reproductive growth. The relative simplicity of the <i>C. elegans</i> nervous system, together with its robust molecular and genetic tool set, make it an ideal system in which to study how environmental stimuli are sensed and integrated to drive developmental plasticity. Food availability, temperature and the presence of dauer pheromone each inform the dauer fate decision. I have taken molecular and genetic approaches to study the mechanism by which pheromone signals are transduced by <i> C. elegans</i> sensory neurons and also to understand how food and pheromone signals are integrated to drive an adaptive developmental choice. I have identified roles in dauer formation for the protein scaffold QUI-1, the rough endoplasmic reticulum protein MACO-1 and a putative Tau tubulin kinase, which we have named PHD-1. I have also identified a critical role for the calcium/calmodulin-dependent protein kinase 1 CMK-1 in encoding the food signal and integrating this information in the dauer fate decision. Furthermore, these studies have led to the previously unrecognized roles for the ASH and AWC neurons in the regulation of dauer formation.</p>

<p> In both mice and humans, males and females exhibit differences in behavior and response to neurological and psychological diseases that are linked to the cortex and hippocampus. The perinatal exposure of males to testosterone secreted by the testes creates alterations in neural structures and behaviors that can persist throughout their lives; however, the molecular mechanisms that underlie the actions of sex steroids to produce these lasting changes are still unclear. Given that regulation of gene expression is a primary mechanism whereby sex steroids exert changes to an organism, I sought to identify genes expressed at different levels between the sexes in the cortex and hippocampus and to determine the effect of testosterone on expression of these genes. Using gene expression microarrays and RT-qPCR, I identified genes that are differentially expressed between the sexes in the neonatal mouse cortex and hippocampus; however, whether perinatal testosterone is regulating these differences remains unclear.</p>

<p> The worm <i>Caenorhabditis elegans</i> is a well-studied model organism in numerous aspects of its biology. This small free living nematode has less than 1,000 cells, but shows clear conservation in both signaling and behavior to mammals in aspects of appetite control. This is of importance to humans, where failure of appetite control is a major factor in the unprecedented obesity epidemic that we see today. </p><p> In general, worm behavior reflects its internal nutritional state and the availability and quality of food. Specifically, worms show a behavioral state that mimics aspects of the mammalian behavioral satiety sequence, which has been termed satiety quiescence. We have used locomotion tracking and Hidden Markov Model analysis to identify worm behavioral state over time, finding quiescence along with the established worm locomotive behaviors roaming and dwelling. Using this analysis as well as more conventional cell biology and genetic approaches we have further investigated satiety signaling pathways. We have found that the neuron ASI is a major center of integration of signals regarding the internal nutritional state of the worms as well as the nutritional content of its environment. Our results show that cGMP causes levels of the TGF&beta; ligand to be increased in fasted worms, which is then released and binds to its receptor on the RIM and RIC neurons. This signaling connects nutritional state to behavioral response, promoting the sleep-like behavioral state satiety quiescence. Additionally, we have begun a candidate approach examining several other groups of signaling molecules for potential roles in satiety quiescence signaling including cannabinoids, multidrug resistance proteins, and neuropeptides. The result of this investigation is a better understanding of mechanisms of satiety quiescence signaling as well as a new tool that provides highly quantitative, unbiased, and automated data to aid in our ongoing work.</p>

<p> Opioids are used for the clinical treatment of pain, but can lead to tolerance and addiction. In this project we examined the role of the serotonin (5-HT) system originating from the dorsal raphe nucleus (DRN) during morphine exposure, withdrawal, abstinence and following an acute stressor capable of initiating behavioral relapse. Following four days of morphine exposure rats showed a preference for the morphine paired side of the conditioned place preference (CPP) chamber. After four days of morphine abstinence, rats showed no net preference for the morphine paired side. The next day rats were exposed to forced swim stress and returned to the CPP chamber where they demonstrated stress-induced reinstatement. Utilizing whole-cell patch-clamp we demonstrated an increase in the amplitude of inhibitory post-synaptic currents (IPSCs) in 5-HT DRN neurons, but not non 5-HT DRN neurons of morphine-conditioned subjects. Next the stress neurohormone corticotrophin releasing factor (CRF) was administered in vitro instead of forced swim. We found an increase in CRF-R2-mediated inward current of 5-HT DRN neurons in animals with a morphine history. From this experiment we concluded that morphine history sensitizes 5-HT DRN neurons to the GABAergic inhibitory effects of stress and to some of the effects of CRF. In the next series of experiments we surgically implanted either morphine or placebo pellets in rats for 72 hours to create physical dependence. The pellets were subsequently removed, and animals experienced up to seven days of abstinence with and without forced swim stress exposure. Real time quantitative PCR was used to measure the mRNA levels of genes at multiple points across this timeline. We examined genes involved in trophic support, stress responses and 5-HT regulation. We determined that mRNA levels for brain-derived neurotrophic factor (BDNF) and the BDNF receptor TrkB were downregulated after opiate exposure, and again following seven days of abstinence. Following seven days of abstinence there was a decrease in mRNA levels of the CRF-R1 receptor and an increase in mRNA levels of the CRF-R2 receptor. During acute opiate exposure there was a decrease in mRNA levels for the autoregulatory 5-HT_1A receptor. Finally following forced swim, there was an increase in mRNA levels of the 5-HT synthesis enzyme TPH2. Collectively these results indicate that a morphine history in abstinent subjects may produce hypofunctioning of the 5-HT DRN system induced by multiple neurochemical mechanisms and this dysregulation may enhance vulnerability to stress-induced relapse.</p>

<p> Ovulation requires neural circuits in the brain to be sequentially exposed to estradiol and progesterone in the female rat. Estradiol-induced neuroprogesterone is essential for the luteinizing hormone (LH) surge and subsequently, ovulation to occur. The LH surge is regulated by gonadotropin-releasing hormone (GnRH) neurons in the diagonal band of Broca (DBB) which do not express progesterone receptors (PR), but are closely associated with anteroventral periventricular nucleus (AVPV) PR-expressing kisspeptin neurons. I tested the hypothesis that estradiol-induced neuroprogesterone activates AVPV kisspeptin neurons to trigger the LH surge. Inhibiting progesterone synthesis blocked estradiol induction of the LH surge that was rescued by subsequent treatment with either progesterone or DBB kisspeptin infusion. Estradiol treatment triggered a robust LH surge that was blocked by AVPV kisspeptin asODN infusion. These results support the hypothesis that neuroprogesterone induces kisspeptin release from AVPV neurons to activate DBB GnRH neurons and trigger the LH surge.</p>

<p> Neural stem cells (NSCs) participate in a delicate balance between maintaining cellular identity through self-renewal and differentiating into myriad neural cell types. Understanding exactly how epigenetic mechanisms regulate this balance and the subsequent differentiation process in adult mammalian brain is an ongoing effort. We conducted a genome wide association study to elucidate the roles of genes in neural progenitors regulated by chromatin modifications. Neural progenitors of baboon SVZ were examined using ChIP-Seq (chromatin immuneprecipitation followed by deep sequencing) to determine genome wide gene targets of three histone modifications: H3K4me3, H3K9me3 and H3K27me3. Our data suggest these chromatin marks are associated with genes responsible for cellular organization and morphology, proliferation and survival, neuron development. Taken together these processes suggest histone modifications, predominantly H3K27me3, are responsible for maintenance of NSC identity. Our findings also highlight the importance of using in vivo models to study the SVZ neurogenic niche and compel examination of the H3K27me3 catalyzing enzyme EZH2. In the future, the role of EZH2 will be determined by EZH2 conditional knockout and overexpression models, using stereotaxic injections of novel Cre protein and lentiviral delivery of EZH2, respectively.</p>